1. Field of the Invention
The invention relates to L-lysine-producing strains of corynebacteria with enhanced pyc gene (pyruvate carboxylase gene), in which strains additional genes, chosen from the group consisting of the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the lysE gene (lysine export carrier gene) and the dapB gene (dihydrodipicolinate reductase gene), but especially the dapA gene, are enhanced and, in particular, over-expressed, and to a process for the preparation of L-lysine.
2. Background Information
L-Lysine is a commercially important L-amino acid which is used especially as a feed additive in animal nutrition. The demand of such feed additives has been steadily increasing in recent years.
L-Lysine is prepared by a fermentation process with L-lysine-producing strains of corynebacteria, especially Corynebacterium glutamicum. Because of the great importance of this product, attempts are constantly being made to improve the preparative process. Improvements to the process may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.
The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites, e.g. S-(2-aminoethyl)cysteine, or auxotrophic for amino acids, e.g. L-leucine, and produce L-lysine.
Methods of recombinant DNA technology have also been used for some years in order to improve L-lysine-producing strains of Corynebacterium glutamicum by amplifying individual biosynthesis genes and studying the effect on the L-lysine production.
Thus, EP-A-0 088 166 reports the increase in productivity, after amplification, of a DNA fragment conferring resistance to aminoethylcysteine. EP-B-0 387 527 reports the increase in productivity, after amplification, of an lysC allele coding for a feedback-resistant aspartate kinase. EP-B-0 197 335 reports the increase in productivity, after amplification, of the dapA gene coding for dihydrodipicolinate synthase. EP-A-0 219 027 reports the increase in productivity, after amplification, of the asd gene coding for aspartate semialdehyde dehydrogenase. Pisabarro et al. (Journal of Bacteriology 175(9), 2743-2749 (1993)) describe the dapB gene coding for dihydrodipicolinate reductase.
The effect of the amplification of primary metabolism genes on the L-lysine production has also been studied. Thus EP-A-0 219 027 reports the increase in productivity, after amplification, of the aspC gene coding for aspartate aminotransferase. EP-B-0 143 195 and EP-B-0 358 940 report the increase in productivity, after amplification, of the ppc gene coding for phosphoenolpyruvate carboxylase. DE-A-198 31 609 reports the increase in productivity, after amplification, of the pyc gene coding for pyruvate carboxylase. The anaplerotic reaction catalyzed by pyruvate carboxylase is of particular importance compared with the reaction catalyzed by phosphoenolpyruvate carboxylase. Thus, Wendisch et al. (FEMS Microbiology Letters 112, 269-274 (1993)) showed that the lysine production of the strain MH20-22B was not impaired by turning off the ppc gene.
Finally, DE-A-195 48 222 describes that an increased activity of the L-lysine export carrier coded for by the lysE gene promotes lysine production.
In addition to these attempts to amplify an individual gene, attempts have also been made to amplify two or more genes simultaneously, thereby improving the L-lysine production in corynebacteria. Thus, DE-A-38 23 451 reports the increase in productivity, after simultaneous amplification, of the asd gene and the dapA gene from Escherichia coli. DE-A-39 43 117 discloses the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase and of the dapA gene. EP-A-0 841 395 particularly reports the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase and of the dapB gene; further improvements could be achieved by additional amplification of the dapB, lysA and ddh genes. EP-A-0 854 189 describes the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase and of the dapA, dapB, lysA and aspC genes. EP-A-0 857 784 particularly reports the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase enzyme and the lysA gene; a further improvement could be achieved by additional amplification of the ppc gene.
It is clear from the many processes described in the state of the art, that there is a need for the development of novel approaches and for the improvement of existing processes for lysine production with corynebacteria.